1. Field of the Invention
The present invention relates generally to the field of regulation of cell growth and proliferative diseases. More specifically, the present invention relates to mutants of the RB-1 and p53 genes and therapeutic uses of such mutants.
2. Description of the Related Art
The control of cell proliferation is a complex process which involves multiple interacting components. Whether a cell grows or not depends on the balance of the expression of negatively-acting and positively-acting growth regulatory genes. Negatively-acting growth regulatory genes are those that, when expressed in or provided to a cell, lead to suppression of cell growth. Positively-acting growth regulatory genes are those which, when expressed in or provided to a cell, stimulate its proliferation.
Recently, several negatively acting growth regulatory genes called tumor suppressor genes which have a negative effect on cell proliferation have been identified. These genes include, but are not limited to, the human retinoblastoma gene, RB-1, and the p53 gene. The absence or inactivation of some of these negative growth regulatory genes has been correlated with certain types of cancer.
The human retinoblastoma gene, RB-1, is the prototype of this class of tumor suppressor genes in which the absence of both alleles of the gene in a cell or the inhibition of the expression of the gene or its gene product will lead to neoplastic or abnormal cellular proliferation. At the molecular level, loss or inactivation of both alleles of RB-1 is involved in the clinical manifestation of tumors such as retinoblastoma and clinically related tumors, such as osteosarcomas, fibrosarcomas, soft tissue sarcomas and melanomas. In addition, loss of the function of RB-1 has also been associated with other types of primary cancer such as primary small cell lung carcinoma, bladder carcinoma, breast carcinomas, cervical carcinomas and prostate carcinomas.
The Tumor Suppressor Genes Rb and p53 suppress the proliferation of cancerous and noncancerous cells
The re-introduction of a wild-type cDNA of RB-1 or p53 have been shown to partially restore normal growth regulation as the re-introduced genes induce growth arrest or retardation in many different tumor cell types. The designation of the Rb gene as a tumor suppression gene stemmed from the fact that inactivation of an allele of the Rb gene is a predisposition to the development of cancer. However, the growth suppression effect of the Rb gene is not restricted to tumor cells. Normal cells which have two copies can be growth arrested or retarded by the introduction of extra copies of the Rb gene under certain growth conditions. Likewise, the ability of a wild type p53 to suppress the growth of noncancerous cells is well documented. Thus, the step controlled by Rb and p53 may not directly affect the tumorigenic phenotype but rather the steps that control the growth of tumor and normal cells alike are affected.
Mechanisms of pathological proliferation of normal cells
There is a wide variety of pathological cell proliferative conditions for which novel methods are needed to provide therapeutic benefits. These pathological conditions may occur in almost all cell types capable of abnormal cell proliferation. Among the cell types which exhibit pathological or abnormal growth are (1) fibroblasts, (2) vascular endothelial cells, and (3) epithelial cells. It can be seen from the above that methods are needed to treat local or disseminated pathological conditions in all or almost all organ and tissue systems of the individual.
For instance, in the eye alone, novel methods may be utilized to treat such a wide variety of pathologic disease states which are due to abnormal proliferation of normal, benign or malignant cells or tissues including, but not limited to, the following: fibroproliferative, vasoproliferative and/or neoplastic diseases of the eye: retinopathy of prematurity, proliferative vitreoretinopathy, proliferative diabetic retinopathy, capillary hemangioma, choroidal neovascular nets, subretinal neovascular nets, senile macular degeneration due to subretinal, neovascularization, corneal neovascularization, macular pucker due to epiretinal membrane proliferation, adult cataracts due to nuclear sclerosis, fibrous ingrowth following trauma or surgery, optic nerve gliomas, angiomatosis retinae, neovascular glaucoma, cavernous hemangioma, rubeosis iridis, sickle cell proliferative retinopathy, epithelial downgrowth after eye surgery or injury, after-cataract membrane, papilloma, retinal neovascularization in thalassemia, subretinal neovascularization due to pseudoxanthoma elasticum, and neurofibromatosis type 1 and 11, retinoblastoma, uveal melanoma, and pseudotumor of the orbit. Other benign cell proliferative diseases for which the present invention is useful include, but are not limited to, psoriasis, ichthyosis, papillomas, basal cell carcinomas, squamous cell carcinoma, and Stevens-Johnson Syndrome.
It should be noted that in the case of normal cells, there are already two normal alleles each of the Rb gene and the P53 gene and yet these wild type proteins fail to prevent the cells from proliferating when the cells were provided growth factors, as for example in tissue culture growth conditions. The uncontrolled proliferation of otherwise resting cells in pathological conditions is also due to the exposure of the cells to growth factors induced by the pathological conditions (see below). For an example, uncontrolled proliferation of blood vessels in the eye in thalassemia can lead to detachment of the retina if the proliferation is not stopped. The introduction of extra copies of the Rb gene or the P53 gene into such cells may or may not be sufficient to suppress the cells from growing. In fact, the introduction of exogenous Rb gene into many tumor cells only retarded the growth rate of the cells instead of complete arrest. The previously reported cell lines such as Saos-2 and DU145 are good examples of this phenomenon. Many more copies of the gene may need to be introduced and it is difficult to control on the one hand the number of copies that need to be or could be introduced. On the other hand, growth factor exposure may lead to inactivation of the expressed Rb proteins.
Regulation of the Activity of Rb Protein by Phosphorylation
If the inactivation of growth suppressor genes is an essential step in the removal of the constraint on cell growth, there must be mechanisms whereby the activities of these gene products are regulated so that a cell may proliferate in a controlled manner. It is conceivable that the expression of a given growth suppressor gene may be regulated quantitatively or qualitatively. In the case of the Rb gene, the ratio of the steady state level of the protein to the cell volume is a constant throughout the cell cycle. This lack of variation of the Rb protein concentration suggests that there must be other mechanisms whereby a cell can regulate the activity of this protein. There are several lines of evidence to show that the activity of the Rb protein (pRb) is regulated by its phosphorylation state. Various growth stimulatory or inhibitory factors exert their effects by perturbing the phosphorylation of growth suppressor gene products such as the Rb protein. Evidence for a role of growth stimulatory factors in the induction of phosphorylation of the Rb proteins have come from the observation that the Rb proteins exist in the underphosphorylated forms in quiescent cells. In addition, when quiescent cells were stimulated to proliferate by exposure either to serum or to growth factors such as EGF together with insulin and transferrin, the predominant form of the Rb protein was underphosphorylated in G1 but became hyperphosphorylated as the cells enter the G1/S boundary and the cells. These data suggest that the Rb protein is a target of the signal transduction pathway induced by serum or growth factors. Finally, senescent human fibroblast cells incapable of responding to the proliferation stimulatory effects of growth factors also failed to phosphorylate the Rb protein.
There are also evidence for a role of growth inhibitory factors in the downregulation of phosphorylation of the Rb proteins. When actively growing leukemia or neuroblastoma cells were treated with retinoic acid or vitamin D3, DMSO other differentiation inducing reagents, the Rb proteins were found to be underphosphorylated prior to the on set of cell growth arrest in the early G1 phase and induced to differentiation. In addition, the treatment of lung epithelial cells with the paracrine growth inhibitory polypeptides-transforming growth factor Beta 1 (TGF.beta.-1) led to the downregulation of phosphorylation of the Rb protein and concomitant arrest of cell growth in late G1. Taken together, these data suggest the underphosphorylated form of the Rb protein actively suppressed cells from traversing the G1/S boundary and that a kinase (S), activated in G1, can inactivate the Rb protein so that the cells may move into the S phase.
While the permanent removal of the Rb gene, by deletion or mutation, in tumor cells allowed growth, removal of the constraint on growth in normal cells having normal expression of the Rb gene is achieved by cell cycle dependent post-translational modification of its gene product, i.e., the Rb protein (pRb). The Rb protein exists in multi-phosphorylated forms and evidence suggests that the underphosphorylated form is the active form. For example, the SV40 large T antigen preferentially binds to the underphosphorylated form of the Rb protein. Phosphorylation of the Rb protein releases it from the SV40 large T antigen and only the underphosphorylated forms of pRb binds to and inhibits transcription from the E2 promotor. Thus, the underphosphorylated form of the Rb protein actively suppresses cell growth, whereas cell proliferation is associated with hyperphosphorylation of the Rb protein. Various growth stimulatory or inhibitory factors may exert their effects by perturbing the phosphorylation of the Rb protein. Therefore, although a normal cell may be expressing the Rb proteins, it can be induced to grow when exposed to appropriate growth factors such as may be found in various growth conditions, including pathological conditions. Under certain pathological conditions, an otherwise normal cell may be induced to proliferate undesirably. Proliferation of normally quiescent fibroblasts in the eyes in diabetic retinopathy is an example of such unwanted induction of cell growth. Left untreated, the proliferating fibroblasts will eventually attach to the retina and lead to its rapid detachment.
Activation of the p53 protein
Similar to the Rb gene, inactivation or mutation of the p53 gene has been frequently observed in tumor and nontumorous cell lines. Wild type p53 protein is also negatively regulated when it binds to viral antigens such as the SV40 large T. The p53 protein functions as a transcription factor and binds to specific DNA sequences. The ability of the wildtype p53 protein to bind DNA is critical for it to function properly. In mutant p53, the ability of the protein to bind to specific DNA target sequences or to activate transcription is lost, suggesting that these activities are importantant for the suppressive function of the protein. However it has also been recently shown that sequence specific DNA binding activity of the p53 protien is cryptic. Newly synthesized p53 protien is innactive in that it cannot bind to its specific DNA sequence unless the protein is modified. One of the sites for modification is amino acid residues 365-393 at the C-terminus. This region is the site for the attachment of an RNA moiety as well as for the binding to the bacterial heat shock proteins dnaK. Binding of an antibody, Pab421, to the 365-389 amino acid residues activates the protein by altering its conformation so that it can bind to its specific DNA sequence.
Difference in the mode of function of pRb and p53
although both Rb and p53 are regarded as tumor suppressor genes, their modes of action are very different. Previously, evidence was provided that these two genes may work on different pathways. Mice null for the Rb gene is embryonically lethal while mice null for p53 develop normally although they are more susceptible to cancer development. At the cellular level, cancer cells that are devoid of p53 can be growth arrested or retarded by the overexpression of the Rb gene even in the absence of exogenous p53. On the other hand, cells devoid of Rb are susceptible to the growth suppression effect of the p53 gene. At the molecular level, p53 and pRb interacts with different protein targets and DNA sequences (directly or indirectly).
The use of the Rb and p53 gene in therapy
While these two proteins are different in their modes of action, they are similar in one aspect, i.e., overexpression of the active forms of the two proteins under experimental conditions lead to the suppression or retardation of growth of cells. Knowing that cell growth is controlled by the expression of suppressor genes such as pRb and p53 and that their proteins need to be in the active conformations in order to function, it is desirable to have active forms of these genes for gene therapy. In the case of pRb, the newly synthesized proteins are underphosphorylated and are active. Inactivation may occur as the cells are exposed to growth factors. It would be desirable to have mutant forms of pRb that would remain in the active form and are resistant to inactivation by phosphorylation. In the case of p53, the newly synthesized protein is inactive in that it cannot bind DNA unless modified. In gene therapy, it would be highly desirable to have mutant forms of the p53 gene, the protein of which is already active even without further modification. Finally, since the two proteins have different modes of action, it would be desirable to use a combination of both the Rb and the p53 genes in therapy such that the mutations or growth conditions that renders a cell resistant to the growth suppression effect of one gene may be susceptible to the other.
The prior art remains deficient in the lack of a tumor suppressor gene mutated so that the gene product (protein) is permanently active and can function even when the cell is exposed to growth factors. More specifically, the prior art is deficient in the lack of effective and functional mutated forms of the p53 and Rb genes. The prior art also remains deficient in the simultaneous application of the wildtype and/or the mutant forms of the Rb and the p53 genes in cancerous and noncancerous cell proliferative diseases.